Cell motility underlies diverse biological processes, both normal and pathological, in the human body including embryonic development, wound healing, development of blood vessels and cancer metastasis. Two major cytoskeletal systems, microtubules and actin, work together to coordinate cell protrusion and retraction during motility. The plus-ends of microtubules enter the cell periphery, target and modulate components of the actin cytoskeleton. However, the signaling mechanisms which functionally link microtubules to actin protrusion are poorly understood. Plus-end tracking proteins selectively bind growing microtubules making them good candidates for mediating the interaction between microtubules and actin. A particular plus- end tracking protein, EB1, is necessary for normal actin protrusion and cell motility in mouse melanoma cells. The research aims and experimental approaches of this proposal are driven by the hypothesis that EB1 protein controls cell motility through regulating actin signaling pathways and interaction with the extracellular matrix. The first aim is to investigate the effect of EB1 depletion on targeting of microtubules to adhesion sites and adhesion site turnover. This aim will involve use of 2-dimensional deconvolution to analyze adhesion site dynamics in live cells. The second and third aims will delineate EB1 position in lamellipodial and filopodial signaling pathways. These aims will be completed using quantitative immunofluorescence microscopy combined with biochemical techniques. The overall goal of the proposed research is to understand the molecular mechanism by which EB1 regulates cell motility. PUBLIC HEALTH RELEVANCE: Spatial control of protrusion during cell motility requires coordinated activities between the microtubule and actin cytoskeleton systems. However, the molecular mechanism that functionally couples microtubules to actin protrusion is poorly understood. The broad goal of this study is to understand the molecular details that link microtubule plus-ends to cell motility relevant to cancer metastasis.